Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53

ABSTRACT

The invention relates to a method wherein a molecule is used for inducing and/or promoting skipping of at least one of exon 43, exon 46, or exons 50-53 of the DMD pre-mRNA in a patient, the method comprising providing the patient with the molecule. The invention also relates to the molecule as such.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/024,558, filed Jun. 29, 2018, which is a continuation of U.S.application Ser. No. 15/289,053, filed Oct. 7, 2016, which is acontinuation of U.S. application Ser. No. 14/631,686, filed Feb. 25,2015, now U.S. Pat. No. 9,499,818, which is a continuation of U.S.application Ser. No. 13/094,571, filed Apr. 26, 2011, which is acontinuation of PCTNL2009/050113, filed Mar. 11, 2009, which is acontinuation-in-part of PCT/NL2008/050673, filed Oct. 27, 2008. Thedisclosures of each of the above-referenced applications areincorporated by reference herein in their entirety.

SEQUENCE LISTING

This specification is being filed with a Sequence Listing in ComputerReadable Form (CFR), which is entitled “0105_07 US1CN4_SL.txt” of 128885bytes in size and was created on Dec. 14, 2020, the content of which isincorporated herein by reference in its entirety.

FIELD

The invention relates to the field of genetics, more specifically humangenetics. The invention in particular relates to modulation of splicingof the human Duchenne Muscular Dystrophy pre-mRNA.

BACKGROUND

Myopathies are disorders that result in functional impairment ofmuscles. Muscular dystrophy (MD) refers to genetic diseases that arecharacterized by progressive weakness and degeneration of skeletalmuscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy(BMD) are the most common childhood forms of muscular dystrophy. Theyare recessive disorders and because the gene responsible for DMD and BMDresides on the X-chromosome, mutations mainly affect males with anincidence of about 1 in 3500 boys.

DMD and BMD are caused by genetic defects in the DMD gene encodingdystrophin, a muscle protein that is required for interactions betweenthe cytoskeleton and the extracellular matrix to maintain muscle fiberstability during contraction. DMD is a severe, lethal neuromusculardisorder resulting in a dependency on wheelchair support before the ageof 12 and DMD patients often die before the age of thirty due torespiratory- or heart failure. In contrast, BMD patients often remainambulatory until later in life, and have near normal life expectancies.DMD mutations in the DMD gene are characterized by frame shiftinginsertions or deletions or nonsense point mutations, resulting in theabsence of functional dystrophin. BMD mutations in general keep thereading frame intact, allowing synthesis of a partly functionaldystrophin.

During the last decade, specific modification of splicing in order torestore the disrupted reading frame of the dystrophin transcript hasemerged as a promising therapy for Duchenne muscular dystrophy (DMD)(van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol Ther. 2008;10(2):140-9, Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84, vanDeutekom et al., N Engl J Med. 2007; 357(26):2677-86).

Using antisense oligonucleotides (AONs) interfering with splicingsignals the skipping of specific exons can be induced in the DMDpre-mRNA, thus restoring the open reading frame and converting thesevere DMD into a milder BMD phenotype (van Deutekom et al. Hum MolGenet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003;12(8):907-14.). In vivo proof-of-concept was first obtained in the mdxmouse model, which is dystrophin-deficient due to a nonsense mutation inexon 23. Intramuscular and intravenous injections of AONs targeting themutated exon 23 restored dystrophin expression for at least three months(Lu et al. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005; 102(1):198-203). This was accompanied by restoration ofdystrophin-associated proteins at the fiber membrane as well asfunctional improvement of the treated muscle. In vivo skipping of humanexons has also been achieved in the hDMD mouse model, which contains acomplete copy of the human DMD gene integrated in chromosome 5 of themouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; ′t Hoenet al. J Biol Chem. 2008; 283: 5899-907).

Recently, a first-in-man study was successfully completed where an AONinducing the skipping of exon 51 was injected into a small area of thetibialis anterior muscle of four DMD patients. Novel dystrophinexpression was observed in the majority of muscle fibers in all fourpatients treated, and the AON was safe and well tolerated (van Deutekomet al. N Engl J Med. 2007; 357: 2677-86).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In human control myotubes, a series of AONs (PS237, PS238, andPS240; SEQ ID NO 65, 66, 16 respectively) targeting exon 43 was testedat 500 nM. PS237 (SEQ ID NO 65) reproducibly induced highest levels ofexon 43 skipping. (M: DNA size marker; NT: non-treated cells)

FIG. 2. In myotubes from a DMD patient with an exon 45 deletion, aseries of AONs (PS177, PS179, PS181, and PS182; SEQ ID NO 91, 70, 110,and 117 respectively) targeting exon 46 was tested at two differentconcentrations (50 and 150 nM). PS182 (SEQ ID NO 117) reproduciblyinduced highest levels of exon 46 skipping. (M: DNA size marker)

FIG. 3. In human control myotubes, a series of AONs (PS245, PS246,PS247, and PS248; SEQ ID NO 167, 165, 166, and 127 respectively)targeting exon 50 was tested at 500 nM. PS248 (SEQ ID NO 127)reproducibly induced highest levels of exon 50 skipping. (M: DNA sizemarker; NT: non-treated cells).

FIG. 4. In human control myotubes, two novel AONs (PS232 and PS236; SEQID NO 246 and 299 respectively) targeting exon 52 were tested at twodifferent concentrations (200 and 500 nM) and directly compared to apreviously described AON (52-1). PS236 (SEQ ID NO 299) reproduciblyinduced highest levels of exon 52 skipping. (M: DNA size marker; NT:non-treated cells).

DETAILED DESCRIPTION Method

In a first aspect, the present invention provides a method for inducing,and/or promoting skipping of at least one of exons 43, 46, 50-53 of theDMD pre-mRNA in a patient, preferably in an isolated cell of a patient,the method comprising providing said cell and/or said patient with amolecule that binds to a continuous stretch of at least 8 nucleotideswithin said exon. It is to be understood that said method encompasses anin vitro, in vivo or ex vivo method.

Accordingly, a method is provided for inducing and/or promoting skippingof at least one of exons 43, 46, 50-53 of DMD pre-mRNA in a patient,preferably in an isolated cell of said patient, the method comprisingproviding said cell and/or said patient with a molecule that binds to acontinuous stretch of at least 8 nucleotides within said exon.

As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMDgene of a DMD or BMD patient.

A patient is preferably intended to mean a patient having DMD or BMD aslater defined herein or a patient susceptible to develop DMD or BMD dueto his or her genetic background. In the case of a DMD patient, anoligonucleotide used will preferably correct one mutation as present inthe DMD gene of said patient and therefore will preferably create a DMDprotein that will look like a BMD protein: said protein will preferablybe a functional dystrophin as later defined herein. In the case of a BMDpatient, an oligonucleotide as used will preferably correct one mutationas present in the BMD gene of said patient and therefore will preferablycreate a dystrophin which will be more functional than the dystrophinwhich was originally present in said BMD patient.

Exon skipping refers to the induction in a cell of a mature mRNA thatdoes not contain a particular exon that is normally present therein.Exon skipping is performed by providing a cell expressing the pre-mRNAof said mRNA with a molecule capable of interfering with essentialsequences such as for example the splice donor of splice acceptorsequence that required for splicing of said exon, or a molecule that iscapable of interfering with an exon inclusion signal that is requiredfor recognition of a stretch of nucleotides as an exon to be included inthe mRNA. The term pre-mRNA refers to a non-processed or partlyprocessed precursor mRNA that is synthesized from a DNA template in thecell nucleus by transcription.

Within the context of the invention, inducing and/or promoting skippingof an exon as indicated herein means that at least 1%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more(muscle) cells of a treated patient will not contain said exon. This ispreferably assessed by PCR as described in the examples.

Preferably, a method of the invention by inducing and/or promotingskipping of at least one of the following exons 43, 46, 50-53 of the DMDpre-mRNA in one or more (muscle) cells of a patient, provides saidpatient with a functional dystrophin protein and/or decreases theproduction of an aberrant dystrophin protein in said patient and/orincreases the production of a functional dystrophin is said patient.

Providing a patient with a functional dystrophin protein and/ordecreasing the production of an aberrant dystrophin protein in saidpatient is typically applied in a DMD patient. Increasing the productionof a functional dystrophin is typically applied in a BMD patient.

Therefore, a preferred method is a method, wherein a patient or one ormore cells of said patient is provided with a functional dystrophinprotein and/or wherein the production of an aberrant dystrophin proteinin said patient is decreased and/or wherein the production of afunctional dystrophin is increased in said patient, wherein the level ofsaid aberrant or functional dystrophin is assessed by comparison to thelevel of said dystrophin in said patient at the onset of the method.

Decreasing the production of an aberrant dystrophin may be assessed atthe mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%,40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrantdystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophinmRNA or protein is also referred to herein as a non-functionaldystrophin mRNA or protein. A non-functional dystrophin protein ispreferably a dystrophin protein which is not able to bind actin and/ormembers of the DGC protein complex. A non-functional dystrophin proteinor dystrophin mRNA does typically not have, or does not encode, adystrophin protein with an intact C-terminus of the protein.

Increasing the production of a functional dystrophin in said patient orin a cell of said patient may be assessed at the mRNA level (by RT-PCRanalysis) and preferably means that a detectable amount of a functionaldystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectabledystrophin mRNA is a functional dystrophin mRNA. Increasing theproduction of a functional dystrophin in said patient or in a cell ofsaid patient may be assessed at the protein level (by immunofluorescenceand western blot analyses) and preferably means that a detectable amountof a functional dystrophin protein is detectable by immunofluorescenceor western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophinprotein is a functional dystrophin protein.

As defined herein, a functional dystrophin is preferably a wild typedystrophin corresponding to a protein having the amino acid sequence asidentified in SEQ ID NO: 1. A functional dystrophin is preferably adystrophin, which has an actin binding domain in its N terminal part(first 240 amino acids at the N terminus), a cysteine-rich domain (aminoacid 3361 till 3685) and a C terminal domain (last 325 amino acids atthe C terminus) each of these domains being present in a wild typedystrophin as known to the skilled person. The amino acids indicatedherein correspond to amino acids of the wild type dystrophin beingrepresented by SEQ ID NO:1. In other words, a functional dystrophin is adystrophin which exhibits at least to some extent an activity of a wildtype dystrophin. “At least to some extent” preferably means at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of acorresponding activity of a wild type functional dystrophin. In thiscontext, an activity of a functional dystrophin is preferably binding toactin and to the dystrophin-associated glycoprotein complex (DGC)(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). Binding of dystrophin to actin and to the DGC complex may bevisualized by either co-immunoprecipitation using total protein extractsor immunofluorescence analysis of cross-sections, from a muscle biopsy,as known to the skilled person.

Individuals or patients suffering from Duchenne muscular dystrophytypically have a mutation in the gene encoding dystrophin that preventsynthesis of the complete protein, i.e of a premature stop prevents thesynthesis of the C-terminus. In Becker muscular dystrophy the DMD genealso comprises a mutation compared to the wild type gene, but themutation does typically not induce a premature stop and the C-terminusis typically synthesized. As a result, a functional dystrophin proteinis synthesized that has at least the same activity in kind as the wildtype protein, not although not necessarily the same amount of activity.The genome of a BMD individual typically encodes a dystrophin proteincomprising the N terminal part (first 240 amino acids at the Nterminus), a cysteine-rich domain (amino acid 3361 till 3685) and a Cterminal domain (last 325 amino acids at the C terminus) but its centralrod shaped domain may be shorter than the one of a wild type dystrophin(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). Exon skipping for the treatment of DMD is typically directedto overcome a premature stop in the pre-mRNA by skipping an exon in therod-shaped domain to correct the reading frame and allow synthesis ofremainder of the dystrophin protein including the C-terminus, albeitthat the protein is somewhat smaller as a result of a smaller roddomain. In a preferred embodiment, an individual having DMD and beingtreated by a method as defined herein will be provided a dystrophinwhich exhibits at least to some extent an activity of a wild typedystrophin. More preferably, if said individual is a Duchenne patient oris suspected to be a Duchenne patient, a functional dystrophin is adystrophin of an individual having BMD: typically said dystrophin isable to interact with both actin and the DGC, but its central rod shapeddomain may be shorter than the one of a wild type dystrophin(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne MuscularDystrophy mutation database: an overview of mutation types andparadoxical cases that confirm the reading-frame rule, Muscle Nerve, 34:135-144). The central rod-shaped domain of wild type dystrophincomprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entriesin the leiden Duchenne Muscular Dystrophy mutation database: an overviewof mutation types and paradoxical cases that confirm the reading-framerule, Muscle Nerve, 34: 135-144). For example, a central rod-shapeddomain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22or 12 to 18 spectrin-like repeats as long as it can bind to actin and toDGC.

A method of the invention may alleviate one or more characteristics of amyogenic or muscle cell of a patient or alleviate one or more symptomsof a DMD patient having a deletion including but not limited to exons44, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53 (correctable by exon 43skipping), 19-45, 21-45, 43-45, 45, 47-54, 47-56 (correctable by exon 46skipping), 51, 51-53, 51-55, 51-57 (correctable by exon 50 skipping),13-50, 19-50, 29-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52(correctable by exon 51 skipping), exons 8-51, 51, 53, 53-55, 53-57,53-59, 53-60, (correctable by exon 52 skipping) and exons 10-52, 42-52,43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52 (correctable by exon 53skipping) in the DMD gene, occurring in a total of 68% of all DMDpatients with a deletion (Aartsma-Rus et al., Hum. Mut. 2009).

Alternatively, a method of the invention may improve one or morecharacteristics of a muscle cell of a patient or alleviate one or moresymptoms of a DMD patient having small mutations in, or single exonduplications of exon 43, 46, 50-53 in the DMD gene, occurring in a totalof 36% of all DMD patients with a deletion (Aartsma-Rus et al, Hum. Mut.2009)

Furthermore, for some patients the simultaneous skipping of one of moreexons in addition to exon 43, exon 46 and/or exon 50-53 is required torestore the open reading frame, including patients with specificdeletions, small (point) mutations, or double or multiple exonduplications, such as (but not limited to) a deletion of exons 44-50requiring the co-skipping of exons 43 and 51, with a deletion of exons46-50 requiring the co-skipping of exons 45 and 51, with a deletion ofexons 44-52 requiring the co-skipping of exons 43 and 53, with adeletion of exons 46-52 requiring the co-skipping of exons 45 and 53,with a deletion of exons 51-54 requiring the co-skipping of exons 50 and55, with a deletion of exons 53-54 requiring the co-skipping of exons 52and 55, with a deletion of exons 53-56 requiring the co-skipping ofexons 52 and 57, with a nonsense mutation in exon 43 or exon 44requiring the co-skipping of exon 43 and 44, with a nonsense mutation inexon 45 or exon 46 requiring the co-skipping of exon 45 and 46, with anonsense mutation in exon 50 or exon 51 requiring the co-skipping ofexon 50 and 51, with a nonsense mutation in exon 51 or exon 52 requiringthe co-skipping of exon 51 and 52, with a nonsense mutation in exon 52or exon 53 requiring the co-skipping of exon 52 and 53, or with a doubleor multiple exon duplication involving exons 43, 46, 50, 51, 52, and/or53.

In a preferred method, the skipping of exon 43 is induced, or theskipping of exon 46 is induced, or the skipping of exon 50 is induced orthe skipping of exon 51 is induced or the skipping of exon 52 is inducedor the skipping of exon 53 is induced. An induction of the skipping oftwo of these exons is also encompassed by a method of the invention. Forexample, preferably skipping of exons 50 and 51, or 52 and 53, or 30 43and 51, or 43 and 53, or 51 and 52. Depending on the type and theidentity (the specific exons involved) of mutation identified in apatient, the skilled person will know which combination of exons needsto be skipped in said patient.

In a preferred method, one or more symptom(s) of a DMD or a BMD patientis/are alleviated and/or one or more characteristic(s) of one or moremuscle cells from a DMD or a BMD patient is/are improved. Such symptomsor characteristics may be assessed at the cellular, tissue level or onthe patient self

An alleviation of one or more characteristics may be assessed by any ofthe following assays on a myogenic cell or muscle cell from a patient:reduced calcium uptake by muscle cells, decreased collagen synthesis,altered morphology, altered lipid biosynthesis, decreased oxidativestress, and/or improved muscle fiber function, integrity, and/orsurvival. These parameters are usually assessed using immunofluorescenceand/or histochemical analyses of cross sections of muscle biopsies.

The improvement of muscle fiber function, integrity and/or survival maybe assessed using at least one of the following assays: a detectabledecrease of creatine kinase in blood, a detectable decrease of necrosisof muscle fibers in a biopsy cross-section of a muscle suspected to bedystrophic, and/or a detectable increase of the homogeneity of thediameter of muscle fibers in a biopsy cross-section of a musclesuspected to be dystrophic. Each of these assays is known to the skilledperson.

Creatine kinase may be detected in blood as described in Hodgetts et al(Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006).A detectable decrease in creatine kinase may mean a decrease of 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to theconcentration of creatine kinase in a same DMD or BMD patient beforetreatment.

A detectable decrease of necrosis of muscle fibers is preferablyassessed in a muscle biopsy, more preferably as described in Hodgetts etal (Hodgetts S., et al (2006), Neuromuscular Disorders, 16:591-602.2006) using biopsy cross-sections. A detectable decrease ofnecrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more of the area wherein necrosis has been identified usingbiopsy cross-sections. The decrease is measured by comparison to thenecrosis as assessed in a same DMD or BMD patient before treatment.

A detectable increase of the homogeneity of the diameter of a musclefiber is preferably assessed in a muscle biopsy cross-section, morepreferably as described in Hodgetts et al (Hodgetts S., et al, (2006),Neuromuscular Disorders, 16: 591-602.2006). The increase is measured bycomparison to the homogeneity of the diameter of a muscle fiber in asame DMD or BMD patient before treatment. An alleviation of one or moresymptoms may be assessed by any of the following assays on the patientself: prolongation of time to loss of walking, improvement of musclestrength, improvement of the ability to lift weight, improvement of thetime taken to rise from the floor, improvement in the nine-meter walkingtime, improvement in the time taken for four-stairs climbing,improvement of the leg function grade, improvement of the pulmonaryfunction, improvement of cardiac function, improvement of the quality oflife. Each of these assays is known to the skilled person. As anexample, the publication of Manzur et al. (Manzur A Y et al, (2008),Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review),Wiley publishers, The Cochrane collaboration.) gives an extensiveexplanation of each of these assays. For each of these assays, as soonas a detectable improvement or prolongation of a parameter measured inan assay has been found, it will preferably mean that one or moresymptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy hasbeen alleviated in an individual using a method of the invention.Detectable improvement or prolongation is preferably a statisticallysignificant improvement or prolongation as described in Hodgetts et al(Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006).Alternatively, the alleviation of one or more symptom(s) of DuchenneMuscular Dystrophy or Becker Muscular Dystrophy may be assessed bymeasuring an improvement of a muscle fiber function, integrity and/orsurvival as later defined herein.

A treatment in a method according to the invention may have a durationof at least one week, at least one month, at least several months, atleast one year, at least 2, 3, 4, 5, 6 years or more.

Each molecule or oligonucleotide or equivalent thereof as defined hereinfor use according to the invention may be suitable for directadministration to a cell, tissue and/or an organ in vivo of individualsaffected by or at risk of developing DMD or BMD, and may be administereddirectly in vivo, ex vivo or in vitro. The frequency of administrationof a molecule or an oligonucleotide or a composition of the inventionmay depend on several parameters such as the age of the patient, themutation of the patient, the number of molecules (dose), the formulationof said molecule. The frequency may be ranged between at least once in atwo weeks, or three weeks or four weeks or five weeks or a longer timeperiod.

A molecule or oligonucleotide or equivalent thereof can be delivered asis to a cell. When administering said molecule, oligonucleotide orequivalent thereof to an individual, it is preferred that it isdissolved in a solution that is compatible with the delivery method. Forintravenous, subcutaneous, intramuscular, intrathecal and/orintraventricular administration it is preferred that the solution is aphysiological salt solution. Particularly preferred for a method of theinvention is the use of an excipient that will further enhance deliveryof said molecule, oligonucleotide or functional equivalent thereof asdefined herein, to a cell and into a cell, preferably a muscle cell.Preferred excipients are defined in the section entitled “pharmaceuticalcomposition”.

In a preferred method of the invention, an additional molecule is usedwhich is able to induce and/or promote skipping of another exon of theDMD pre-mRNA of a patient. Preferably, the second exon is selected from:exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62,63, 65, 66, 69, or 75 of the DMD pre-mRNA of a patient. Molecules whichcan be used are depicted in any one of Table 1 to 7. This way, inclusionof two or more exons of a DMD pre-mRNA in mRNA produced from thispre-mRNA is prevented. This embodiment is further referred to as double-or multi-exon skipping (Aartsma-Rus A, Janson A A, Kaman W E, et al.Antisense-induced multiexon skipping for Duchenne muscular dystrophymakes more sense. Am J Hum Genet 2004; 74(1):83-92, Aartsma-Rus A, KamanW E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. Exploringthe frontiers of therapeutic exon skipping for Duchenne musculardystrophy by double targeting within one or multiple exons. Mol Ther2006; 14(3):401-7). In most cases double-exon skipping results in theexclusion of only the two targeted exons from the DMD pre-mRNA. However,in other cases it was found that the targeted exons and the entireregion in between said exons in said pre-mRNA were not present in theproduced mRNA even when other exons (intervening exons) were present insuch region. This multi-skipping was notably so for the combination ofoligonucleotides derived from the DMD gene, wherein one oligonucleotidefor exon 45 and one oligonucleotide for exon 51 was added to a celltranscribing the DMD gene. Such a set-up resulted in mRNA being producedthat did not contain exons 45 to 51. Apparently, the structure of thepre-mRNA in the presence of the mentioned oligonucleotides was such thatthe splicing machinery was stimulated to connect exons 44 and 52 to eachother.

It is possible to specifically promote the skipping of also theintervening exons by providing a linkage between the two complementaryoligonucleotides. Hence, in one embodiment stretches of nucleotidescomplementary to at least two dystrophin exons are separated by alinking moiety. The at least two stretches of nucleotides are thuslinked in this embodiment so as to form a single molecule.

In case, more than one compounds or molecules are used in a method ofthe invention, said compounds can be administered to an individual inany order. In one embodiment, said compounds are administeredsimultaneously (meaning that said compounds are administered within 10hours, preferably within one hour). This is however not necessary. Inanother embodiment, said compounds are administered sequentially.

Molecule

In a second aspect, there is provided a molecule for use in a method asdescribed in the previous section entitled “Method”. A molecule asdefined herein is preferably an oligonucleotide or antisenseoligonucleotide (AON).

It was found by the present investigators that any of exon 43, 46, 50-53is specifically skipped at a high frequency using a molecule thatpreferably binds to a continuous stretch of at least 8 nucleotideswithin said exon. Although this effect can be associated with a higherbinding affinity of said molecule, compared to a molecule that binds toa continuous stretch of less than 8 nucleotides, there could be otherintracellular parameters involved that favor thermodynamic, kinetic, orstructural characteristics of the hybrid duplex. In a preferredembodiment, a molecule that binds to a continuous stretch of at least 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50 nucleotides within said exon is used.

In a preferred embodiment, a molecule or an oligonucleotide of theinvention which comprises a sequence that is complementary to a part ofany of exon 43, 46, 50-53 of DMD pre-mRNA is such that the complementarypart is at least 50% of the length of the oligonucleotide of theinvention, more preferably at least 60%, even more preferably at least70%, even more preferably at least 80%, even more preferably at least90% or even more preferably at least 95%, or even more preferably 98%and most preferably up to 100%. “A part of said exon” preferably means astretch of at least 8 nucleotides. In a most preferred embodiment, anoligonucleotide of the invention consists of a sequence that iscomplementary to part of said exon DMD pre-mRNA as defined herein. Forexample, an oligonucleotide may comprise a sequence that iscomplementary to part of said exon DMD pre-mRNA as defined herein andadditional flanking sequences. In a more preferred embodiment, thelength of said complementary part of said oligonucleotide is of at least8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 nucleotides. Preferably, additional flankingsequences are used to modify the binding of a protein to said moleculeor oligonucleotide, or to modify a thermodynamic property of theoligonucleotide, more preferably to modify target RNA binding affinity.

A preferred molecule to be used in a method of the invention binds or iscomplementary to a continuous stretch of at least 8 nucleotides withinone of the following nucleotide sequences selected from:

(SEQ ID NO: 2) 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ for skipping of exon 43;(SEQ ID NO: 3) 5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ for skipping of exon 46; (SEQ ID NO: 4)5′-GGCGGUAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUGACCUAGCUCCUGGACUGACCACUAUUGG-3′ for skipping of exon 50; (SEQ ID NO: 5)5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAGGAAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUGUUGGAGGU AC-3′for skipping of exon 51; (SEQ ID NO: 6)5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′for skipping of exon 52; and (SEQ ID NO: 7)5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ for skipping of exon 53.

Of the numerous molecules that theoretically can be prepared to bind tothe continuous nucleotide stretches as defined by SEQ ID NO 2-7 withinone of said exons, the invention provides distinct molecules that can beused in a method for efficiently skipping of at least one of exon 43,exon 46 and/or exon 50-53. Although the skipping effect can be addressedto the relatively high density of putative SR protein binding siteswithin said stretches, there could be other parameters involved thatfavor uptake of the molecule or other, intracellular parameters such asthermodynamic, kinetic, or structural characteristics of the hybridduplex.

It was found that a molecule that binds to a continuous stretchcomprised within or consisting of any of SEQ ID NO 2-7 results in highlyefficient skipping of exon 43, exon 46 and/or exon 50-53 respectively ina cell and/or in a patient provided with this molecule. Therefore, in apreferred embodiment, a method is provided wherein a molecule binds to acontinuous stretch of at least 8, 10, 12, 15, 18, 20, 25, 30, 35, 40,45, 50 nucleotides within SEQ ID NO 2-7.

In a preferred embodiment for inducing and/or promoting the skipping ofany of exon 43, exon 46 and/or exon 50-53, the invention provides amolecule comprising or consisting of an antisense nucleotide sequenceselected from the antisense nucleotide sequences depicted in any ofTables 1 to 6. A molecule of the invention preferably comprises orconsist of the antisense nucleotide sequence of SEQ ID NO 16, SEQ ID NO65, SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 117, SEQ ID NO127, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 246, SEQ IDNO 299, SEQ ID NO:357.

A preferred molecule of the invention comprises a nucleotide-based ornucleotide or an antisense oligonucleotide sequence of between 8 and 50nucleotides or bases, more preferred between 10 and 50 nucleotides, morepreferred between 20 and 40 nucleotides, more preferred between 20 and30 nucleotides, such as 20 nucleotides, 21 nucleotides, 22 nucleotides,23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides. A mostpreferred molecule of the invention comprises a nucleotide-basedsequence of 25 nucleotides.

Furthermore, none of the indicated sequences is derived from conservedparts of splice-junction sites. Therefore, said molecule is not likelyto mediate differential splicing of other exons from the DMD pre-mRNA orexons from other genes.

In one embodiment, a molecule of the invention is a compound moleculethat binds to the specified sequence, or a protein such as anRNA-binding protein or a non-natural zinc-finger protein that has beenmodified to be able to bind to the corresponding nucleotide sequence ona DMD pre-RNA molecule. Methods for screening compound molecules thatbind specific nucleotide sequences are, for example, disclosed inPCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are hereinincorporated by reference. Methods for designing RNA-binding Zinc-fingerproteins that bind specific nucleotide sequences are disclosed byFriesen and Darby, Nature Structural Biology 5: 543-546 (1998) which isherein incorporated by reference.

A preferred molecule of the invention binds to at least part of thesequence of SEQ ID NO 2: 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ which is present in exon 43of the DMD gene. More preferably, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 8 to SEQ ID NO 69.

In an even more preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO:16 and/or SEQ ID NO:65. In a most preferred embodiment, the inventionprovides a molecule comprising or consisting of the antisense nucleotidesequence of SEQ ID NO 65. It was found that this molecule is veryefficient in modulating splicing of exon 43 of the DMD pre-mRNA in amuscle cell and/or in a patient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 3:5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ which is present in exon 46 of theDMD gene. More preferably, the invention provides a molecule comprisingor consisting of the antisense nucleotide sequence of SEQ ID NO 70 toSEQ ID NO 122.

In an even more preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 70, SEQ ID NO 91, SEQ ID NO 110, and/or SEQ ID N0117.

In a most preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 117. It was found that this molecule is very efficient in modulatingsplicing of exon 46 of the DMD pre-mRNA in a muscle cell or in apatient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 4: 5′-GGCGGUAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUG ACCUAGCUCCUGGACUGACCACUAUUGG-3′ which is present inexon 50 of the DMD gene. More preferably, the invention provides amolecule comprising or consisting of the antisense nucleotide sequenceof SEQ ID NO 123 to SEQ ID NO 167 and/or SEQ ID NO 529 to SEQ ID NO 535.

In an even more preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 127, or SEQ ID NO 165, or SEQ ID NO 166 and/or SEQ ID NO 167.

In a most preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 127. It was found that this molecule is very efficient in modulatingsplicing of exon 50 of the DMD pre-mRNA in a muscle cell and/or in apatient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 5:5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAGGAAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3′ which ispresent in exon 51 of the DMD gene. More preferably, the inventionprovides a molecule comprising or consisting of the antisense nucleotidesequence of SEQ ID NO 168 to SEQ ID NO 241.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 6:5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′ which is present inexon 52 of the DMD gene. More preferably, the invention provides amolecule comprising or consisting of the antisense nucleotide sequenceof SEQ ID NO 242 to SEQ ID NO 310. In an even more preferred embodiment,the invention provides a molecule comprising or consisting of theantisense nucleotide sequence of SEQ ID NO 246 and/or SEQ ID NO 299. Ina most preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 299. It was found that this molecule is very efficient in modulatingsplicing of exon 52 of the DMD pre-mRNA in a muscle cell and/or in apatient.

Another preferred molecule of the invention binds to at least part ofthe sequence of SEQ ID NO 7:5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ which is present in exon 53 of the DMDgene. More preferably, the invention provides a molecule comprising orconsisting of the antisense nucleotide sequence of SEQ ID NO 311 to SEQID NO 358.

In a most preferred embodiment, the invention provides a moleculecomprising or consisting of the antisense nucleotide sequence of SEQ IDNO 357. It was found that this molecule is very efficient in modulatingsplicing of exon 53 of the DMD pre-mRNA in a muscle cell and/or in apatient.

A nucleotide sequence of a molecule of the invention may contain RNAresidues, or one or more DNA residues, and/or one or more nucleotideanalogues or equivalents, as will be further detailed herein below.

It is preferred that a molecule of the invention comprises one or moreresidues that are modified to increase nuclease resistance, and/or toincrease the affinity of the antisense nucleotide for the targetsequence. Therefore, in a preferred embodiment, the antisense nucleotidesequence comprises at least one nucleotide analogue or equivalent,wherein a nucleotide analogue or equivalent is defined as a residuehaving a modified base, and/or a modified backbone, and/or a non-naturalinternucleoside linkage, or a combination of these modifications.

In a preferred embodiment, the nucleotide analogue or equivalentcomprises a modified backbone. Examples of such backbones are providedby morpholino backbones, carbamate backbones, siloxane backbones,sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetylbackbones, methyleneformacetyl backbones, riboacetyl backbones, alkenecontaining backbones, sulfamate, sulfonate and sulfonamide backbones,methyleneimino and methylenehydrazino backbones, and amide backbones.Phosphorodiamidate morpholino oligomers are modified backboneoligonucleotides that have previously been investigated as antisenseagents. Morpholino oligonucleotides have an uncharged backbone in whichthe deoxyribose sugar of DNA is replaced by a six membered ring and thephosphodiester linkage is replaced by a phosphorodiamidate linkage.Morpholino oligonucleotides are resistant to enzymatic degradation andappear to function as antisense agents by arresting translation orinterfering with pre-mRNA splicing rather than by activating RNase H.Morpholino oligonucleotides have been successfully delivered to tissueculture cells by methods that physically disrupt the cell membrane, andone study comparing several of these methods found that scrape loadingwas the most efficient method of delivery; however, because themorpholino backbone is uncharged, cationic lipids are not effectivemediators of morpholino oligonucleotide uptake in cells. A recent reportdemonstrated triplex formation by a morpholino oligonucleotide and,because of the non-ionic backbone, these studies showed that themorpholino oligonucleotide was capable of triplex formation in theabsence of magnesium.

It is further preferred that that the linkage between the residues in abackbone do not include a phosphorus atom, such as a linkage that isformed by short chain alkyl or cycloalkyl internucleoside linkages,mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, orone or more short chain heteroatomic or heterocyclic internucleosidelinkages.

A preferred nucleotide analogue or equivalent comprises a PeptideNucleic Acid (PNA), having a modified polyamide backbone (Nielsen, etal. (1991) Science 254, 1497-1500). PNA-based molecules are true mimicsof DNA molecules in terms of base-pair recognition. The backbone of thePNA is composed of N-(2-aminoethyl)-glycine units linked by peptidebonds, wherein the nucleobases are linked to the backbone by methylenecarbonyl bonds. An alternative backbone comprises a one-carbon extendedpyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun,495-497). Since the backbone of a PNA molecule contains no chargedphosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNAor RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365,566-568).

A further preferred backbone comprises a morpholino nucleotide analog orequivalent, in which the ribose or deoxyribose sugar is replaced by a6-membered morpholino ring. A most preferred nucleotide analog orequivalent comprises a phosphorodiamidate morpholino oligomer (PMO), inwhich the ribose or deoxyribose sugar is replaced by a 6-memberedmorpholino ring, and the anionic phosphodiester linkage between adjacentmorpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent of theinvention comprises a substitution of one of the non-bridging oxygens inthe phosphodiester linkage. This modification slightly destabilizesbase-pairing but adds significant resistance to nuclease degradation. Apreferred nucleotide analogue or equivalent comprises phosphorothioate,chiral phosphorothioate, phosphorodithioate, phosphotriester,aminoalkylphosphotriester, H-phosphonate, methyl and otheralkylphosphonate including 3′-alkylene phosphonate, 5′-alkylenephosphonate and chiral phosphonate, phosphinate, phosphoramidateincluding 3′-amino phosphoramidate and aminoalkylphosphoramidate,thionophosphoramidate, thionoalkylphosphonate,thionoalkylphosphotriester, selenophosphate or boranophosphate.

A further preferred nucleotide analogue or equivalent of the inventioncomprises one or more sugar moieties that are mono- or disubstituted atthe 2′, 3′ and/or 5′ position such as a —OH; —F; substituted orunsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl,alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted byone or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy,-aminopropoxy; -aminoxy; methoxyethoxy; -dimethylaminooxyethoxy; and-dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose orderivative thereof, or a deoxypyranose or derivative thereof, preferablya ribose or a derivative thereof, or a deoxyribose or a derivativethereof. Such preferred derivatized sugar moieties comprise LockedNucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or4′ carbon atom of the sugar ring thereby forming a bicyclic sugarmoiety. A preferred LNA comprises 2′-0,4′-C-ethylene-bridged nucleicacid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242).These substitutions render the nucleotide analogue or equivalent RNase Hand nuclease resistant and increase the affinity for the target RNA.

It is understood by a skilled person that it is not necessary for allpositions in an antisense oligonucleotide to be modified uniformly. Inaddition, more than one of the aforementioned analogues or equivalentsmay be incorporated in a single antisense oligonucleotide or even at asingle position within an antisense oligonucleotide. In certainembodiments, an antisense oligonucleotide of the invention has at leasttwo different types of analogues or equivalents.

A preferred antisense oligonucleotide according to the inventioncomprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, suchas 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose,2′-O-propyl modified ribose, and/or substituted derivatives of thesemodifications such as halogenated derivatives.

A most preferred antisense oligonucleotide according to the inventioncomprises of 2′-O-methyl phosphorothioate ribose.

A functional equivalent of a molecule of the invention may be defined asan oligonucleotide as defined herein wherein an activity of saidfunctional equivalent is retained to at least some extent. Preferably,an activity of said functional equivalent is inducing exon 43, 46, 50,51, 52, or 53 skipping and providing a functional dystrophin protein.Said activity of said functional equivalent is therefore preferablyassessed by detection of exon 43, 46, 50, 51, 52, or 53 skipping and byquantifying the amount of functional dystrophin protein. A functionaldystrophin is herein preferably defined as being a dystrophin able tobind actin and members of the DGC protein complex. The assessment ofsaid activity of an oligonucleotide is preferably done by RT-PCR or byimmunofluorescence or Western blot analyses. Said activity is preferablyretained to at least some extent when it represents at least 50%, or atleast 60%, or at least 70% or at least 80% or at least 90% or at least95% or more of corresponding activity of said oligonucleotide thefunctional equivalent derives from. Throughout this application, whenthe word oligonucleotide is used it may be replaced by a functionalequivalent thereof as defined herein.

It will be understood by a skilled person that distinct antisenseoligonucleotides can be combined for efficiently skipping any of exon43, exon 46, exon 50, exon 51, exon 52 and/or exon 53 of the human DMDpre-mRNA. It is encompassed by the present invention to use one, two,three, four, five or more oligonucleotides for skipping one of saidexons (i.e., exon, 43, 46, 50, 51, 52, or 53). It is also encompassed touse at least two oligonucleotides for skipping at least two, of saidexons. Preferably two of said exons are skipped. More preferably, thesetwo exons are:

−43 and 51, or−43 and 53, or−50 and 51, or−51 and 52, or−52 and 53.

The skilled person will know which combination of exons is preferred tobe skipped depending on the type, the number and the location of themutation present in a DMD or BMD patient.

An antisense oligonucleotide can be linked to a moiety that enhancesuptake of the antisense oligonucleotide in cells, preferably musclecells. Examples of such moieties are cholesterols, carbohydrates,vitamins, biotin, lipids, phospholipids, cell-penetrating peptidesincluding but not limited to antennapedia, TAT, transportan andpositively charged amino acids such as oligoarginine, poly-arginine,oligolysine or polylysine, antigen-binding domains such as provided byan antibody, a Fab fragment of an antibody, or a single chain antigenbinding domain such as a cameloid single domain antigen-binding domain.

A preferred antisense oligonucleotide comprises a peptide-linked PMO.

A preferred antisense oligonucleotide comprising one or more nucleotideanalogs or equivalents of the invention modulates splicing in one ormore muscle cells, including heart muscle cells, upon systemic delivery.In this respect, systemic delivery of an antisense oligonucleotidecomprising a specific nucleotide analog or equivalent might result intargeting a subset of muscle cells, while an antisense oligonucleotidecomprising a distinct nucleotide analog or equivalent might result intargeting of a different subset of muscle cells. Therefore, in oneembodiment it is preferred to use a combination of antisenseoligonucleotides comprising different nucleotide analogs or equivalentsfor inducing skipping of exon 43, 46, 50, 51, 52, or 53 of the human DMDpre-mRNA.

A cell can be provided with a molecule capable of interfering withessential sequences that result in highly efficient skipping of exon 43,exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNAby plasmid-derived antisense oligonucleotide expression or viralexpression provided by adenovirus- or adeno-associated virus-basedvectors. In a preferred embodiment, there is provided a viral-basedexpression vector comprising an expression cassette that drivesexpression of a molecule as identified herein. Expression is preferablydriven by a polymerase III promoter, such as a U1, a U6, or a U7 RNApromoter. A muscle or myogenic cell can be provided with a plasmid forantisense oligonucleotide expression by providing the plasmid in anaqueous solution. Alternatively, a plasmid can be provided bytransfection using known transfection agentia such as, for example,LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500(MBI Fermentas)), or derivatives thereof.

One preferred antisense oligonucleotide expression system is anadenovirus associated virus (AAV)-based vector. Single chain and doublechain AAV-based vectors have been developed that can be used forprolonged expression of small antisense nucleotide sequences for highlyefficient skipping of exon 43, 46, 50, 51, 52 or 53 of the DMD pre-mRNA.

A preferred AAV-based vector comprises an expression cassette that isdriven by a polymerase III-promoter (Pol III). A preferred Pol IIIpromoter is, for example, a Ul, a U6, or a U7 RNA promoter.

The invention therefore also provides a viral-based vector, comprising aPol III-promoter driven expression cassette for expression of one ormore antisense sequences of the invention for inducing skipping of exon43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMDpre-mRNA.

Pharmaceutical Composition

If required, a molecule or a vector expressing an antisenseoligonucleotide of the invention can be incorporated into apharmaceutically active mixture or composition by adding apharmaceutically acceptable carrier.

Therefore, in a further aspect, the invention provides a composition,preferably a pharmaceutical composition comprising a molecule comprisingan antisense oligonucleotide according to the invention, and/or aviral-based vector expressing the antisense sequence(s) according to theinvention and a pharmaceutically acceptable carrier.

A preferred pharmaceutical composition comprises a molecule as definedherein and/or a vector as defined herein, and a pharmaceuticalacceptable carrier or excipient, optionally combined with a moleculeand/or a vector as defined herein which is able to induce skipping ofexon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62,63, 65, 66, 69, or 75 of the DMD pre-mRNA. Preferred molecules able toinduce skipping of any of these exon are identified in any one of Tables1 to 7.

Preferred excipients include excipients capable of forming complexes,vesicles and/or liposomes that deliver such a molecule as definedherein, preferably an oligonucleotide complexed or trapped in a vesicleor liposome through a cell membrane. Many of these excipients are knownin the art. Suitable excipients comprise polyethylenimine andderivatives, or similar cationic polymers, including polypropyleneimineor polyethylenimine copolymers (PECs) and derivatives, ExGen 500,synthetic amphiphils (SAINT-18), lipofectin, DOTAP and/or viral capsidproteins that are capable of self-assembly into particles that candeliver such molecule, preferably an oligonucleotide as defined hereinto a cell, preferably a muscle cell. Such excipients have been shown toefficiently deliver (oligonucleotide such as antisense) nucleic acids toa wide variety of cultured cells, including muscle cells. Their hightransfection potential is combined with an excepted low to moderatetoxicity in terms of overall cell survival. The ease of structuralmodification can be used to allow further modifications and the analysisof their further (in vivo) nucleic acid transfer characteristics andtoxicity.

Lipofectin represents an example of a liposomal transfection agent. Itconsists of two lipid components, a cationic lipid N-[1-(2,3dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAPwhich is the methylsulfate salt) and a neutral lipiddioleoylphosphatidylethanolamine (DOPE). The neutral component mediatesthe intracellular release. Another group of delivery systems arepolymeric nanoparticles.

Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, whichare well known as DNA transfection reagent can be combined withbutylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulatecationic nanoparticles that can deliver a molecule or a compound asdefined herein, preferably an oligonucleotide across cell membranes intocells.

In addition to these common nanoparticle materials, the cationic peptideprotamine offers an alternative approach to formulate a compound asdefined herein, preferably an oligonucleotide as colloids. Thiscolloidal nanoparticle system can form so called proticles, which can beprepared by a simple self-assembly process to package and mediateintracellular release of a compound as defined herein, preferably anoligonucleotide. The skilled person may select and adapt any of theabove or other commercially available alternative excipients anddelivery systems to package and deliver a compound as defined herein,preferably an oligonucleotide for use in the current invention todeliver said compound for the treatment of Duchenne Muscular Dystrophyor Becker Muscular Dystrophy in humans.

In addition, a compound as defined herein, preferably an oligonucleotidecould be covalently or non-covalently linked to a targeting ligandspecifically designed to facilitate the uptake into the cell, cytoplasmand/or its nucleus. Such ligand could comprise (i) a compound (includingbut not limited to peptide(-like) structures) recognizing cell, tissueor organ specific elements facilitating cellular uptake and/or (ii) achemical compound able to facilitate the uptake into cells and/or theintracellular release of a compound as defined herein, preferably anoligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, a compound as defined herein,preferably an oligonucleotide are formulated in a medicament which isprovided with at least an excipient and/or a targeting ligand fordelivery and/or a delivery device of said compound to a cell and/orenhancing its intracellular delivery. Accordingly, the invention alsoencompasses a pharmaceutically acceptable composition comprising acompound as defined herein, preferably an oligonucleotide and furthercomprising at least one excipient and/or a targeting ligand for deliveryand/or a delivery device of said compound to a cell and/or enhancing itsintracellular delivery. It is to be understood that a molecule orcompound or oligonucleotide may not be formulated in one singlecomposition or preparation. Depending on their identity, the skilledperson will know which type of formulation is the most appropriate foreach compound.

In a preferred embodiment, an in vitro concentration of a molecule or anoligonucleotide as defined herein, which is ranged between 0.1 nM and 1μM is used. More preferably, the concentration used is ranged between0.3 to 400 nM, even more preferably between 1 to 200 nM. A molecule oran oligonucleotide as defined herein may be used at a dose which isranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If severalmolecules or oligonucleotides are used, these concentrations may referto the total concentration of oligonucleotides or the concentration ofeach oligonucleotide added. The ranges of concentration ofoligonucleotide(s) as given above are preferred concentrations for invitro or ex vivo uses. The skilled person will understand that dependingon the oligonucleotide(s) used, the target cell to be treated, the genetarget and its expression levels, the medium used and the transfectionand incubation conditions, the concentration of oligonucleotide(s) usedmay further vary and may need to be optimized any further.

More preferably, a compound preferably an oligonucleotide to be used inthe invention to prevent, treat DMD or BMD are synthetically producedand administered directly to a cell, a tissue, an organ and/or patientsin formulated form in a pharmaceutically acceptable composition orpreparation. The delivery of a pharmaceutical composition to the subjectis preferably carried out by one or more parenteral injections, e.g.,intravenous and/or subcutaneous and/or intramuscular and/or intrathecaland/or intraventricular administrations, preferably injections, at oneor at multiple sites in the human body.

A preferred oligonucleotide as defined herein optionally comprising oneor more nucleotide analogs or equivalents of the invention modulatessplicing in one or more muscle cells, including heart muscle cells, uponsystemic delivery. In this respect, systemic delivery of anoligonucleotide comprising a specific nucleotide analog or equivalentmight result in targeting a subset of muscle cells, while anoligonucleotide comprising a distinct nucleotide analog or equivalentmight result in targeting of a different subset of muscle cells.

In this respect, systemic delivery of an oligonucleotide comprising aspecific nucleotide analog or equivalent might result in targeting asubset of muscle cells, while an oligonucleotide comprising a distinctnucleotide analog or equivalent might result in targeting a differentsubset of muscle cells. Therefore, in this embodiment, it is preferredto use a combination of oligonucleotides comprising different nucleotideanalogs or equivalents for modulating splicing of the DMD mRNA in atleast one type of muscle cells.

In a preferred embodiment, there is provided a molecule or a viral-basedvector for use as a medicament, preferably for modulating splicing ofthe DMD pre-mRNA, more preferably for promoting or inducing skipping ofany of exon 43, 46, 50-53 as identified herein.

Use

In yet a further aspect, the invention provides the use of an antisenseoligonucleotide or molecule according to the invention, and/or aviral-based vector that expresses one or more antisense sequencesaccording to the invention and/or a pharmaceutical composition, formodulating splicing of the DMD pre-mRNA. The splicing is preferablymodulated in a human myogenic cell or muscle cell in vitro. Morepreferred is that splicing is modulated in a human muscle cell in vivo.Accordingly, the invention further relates to the use of the molecule asdefined herein and/or the vector as defined herein and/or or thepharmaceutical composition as defined herein for modulating splicing ofthe DMD pre-mRNA or for the preparation of a medicament for thetreatment of a DMD or BMD patient.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, the verb “to consist” may be replaced by“to consist essentially of’ meaning that a molecule or a viral-basedvector or a composition as defined herein may comprise additionalcomponent(s) than the ones specifically identified, said additionalcomponent(s) not altering the unique characteristic of the invention. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the element ispresent, unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”. Each embodiment as identified herein may becombined together unless otherwise indicated. All patent and literaturereferences cited in the present specification are hereby incorporated byreference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

EXAMPLES Examples 1-4 Materials and Methods

AON design was based on (partly) overlapping open secondary structuresof the target exon RNA as predicted by them-fold program, on (partly)overlapping putative SR— protein binding sites as predicted by theESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V.(Leiden, Netherlands), and contain 2′-O-methyl RNA and full-lengthphosphorothioate (PS) backbones.

Tissue Culturing, Transfection and RT-PCR Analysis

Myotube cultures derived from a healthy individual (“human control”)(examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patientcarrying an exon 45 deletion (example 2; exon 46 skipping) wereprocessed as described previously (Aartsma-Rus et al., Neuromuscul.Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For thescreening of AONs, myotube cultures were transfected with 50 nM and 150nM (example 2), 200 nM and 500 nM (example 4) or 500 nM only (examples 1and 3) of each AON. Transfection reagent UNIFectylin (ProsensaTherapeutics BV, Netherlands) was used, with 2 μl UNIFectylin per AON.Exon skipping efficiencies were determined by nested RT-PCR analysisusing primers in the exons flanking the targeted exons (43, 46, 50, 51,52, or 53). PCR fragments were isolated from agarose gels for sequenceverification. For quantification, the PCR products were analyzed usingthe DNA 1000 LabChips Kit on the Agilent 2100 bioanalyzer (AgilentTechnologies, USA).

Results DMD Exon 43 Skipping.

A series of AONs targeting sequences within exon 43 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 43 herein definedas SEQ ID NO 2, was indeed capable of inducing exon 43 skipping. PS237(SEQ ID NO: 65) reproducibly induced highest levels of exon 43 skipping(up to 66%) at 500 nM, as shown in FIG. 1. For comparison, also PS238and PS240 are shown, inducing exon 43 skipping levels up to 13% and 36%respectively (FIG. 1). The precise skipping of exon 43 was confirmed bysequence analysis of the novel smaller transcript fragments. No exon 43skipping was observed in non-treated cells (NT).

DMD Exon 46 Skipping.

A series of AONs targeting sequences within exon 46 were designed andtransfected in myotube cultures derived from a DMD patient carrying anexon 45 deletion in the DMD gene. For patients with such mutationantisense-induced exon 46 skipping would induce the synthesis of anovel, BMD-like dystrophin protein that may indeed alleviate one or moresymptoms of the disease. Subsequent RT-PCR and sequence analysis ofisolated RNA demonstrated that almost all AONs targeting a continuousnucleotide stretch within exon 46 herein defined as SEQ ID NO 3, wasindeed capable of inducing exon 46 skipping, even at relatively low AONconcentrations of 50 nM. PS182 (SEQ ID NO: 117) reproducibly inducedhighest levels of exon 46 skipping (up to 50% at 50 nM and 74% at 150nM), as shown in FIG. 2. For comparison, also PS177, PS179, and PS181are shown, inducing exon 46 skipping levels up to 55%, 58% and 42%respectively at 150 nM (FIG. 2). The precise skipping of exon 46 wasconfirmed by sequence analysis of the novel smaller transcriptfragments. No exon 46 skipping was observed in non-treated cells (NT).

DMD Exon 50 Skipping.

A series of AONs targeting sequences within exon 50 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 50 herein definedas SEQ ID NO 4, was indeed capable of inducing exon 50 skipping. PS248(SEQ ID NO: 127) reproducibly induced highest levels of exon 50 skipping(up to 35% at 500 nM), as shown in FIG. 3. For comparison, also PS245,PS246, and PS247 are shown, inducing exon 50 skipping levels up to14-16% at 500 nM (FIG. 3). The precise skipping of exon 50 was confirmedby sequence analysis of the novel smaller transcript fragments. No exon50 skipping was observed in non-treated cells (NT).

DMD Exon 51 Skipping

A series of AONs targeting sequences within exon 51 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 51 herein definedas SEQ ID NO 5, was indeed capable of inducing exon 51 skipping. The AONwith SEQ ID NO 180 reproducibly induced highest levels of exon 51skipping (not shown).

DMD Exon 52 Skipping.

A series of AONs targeting sequences within exon 52 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 52 herein definedas SEQ ID NO 6, was indeed capable of inducing exon 52 skipping. PS236(SEQ ID NO: 299) reproducibly induced highest levels of exon 52 skipping(up to 88% at 200 nM and 91% at 500 nM), as shown in FIG. 4. Forcomparison, also PS232 and AON 52-1 (previously published by Aartsma-Ruset al. Oligonucleotides 2005) are shown, inducing exon 52 skipping atlevels up to 59% and 10% respectively when applied at 500 nM (FIG. 4).The precise skipping of exon 52 was confirmed by sequence analysis ofthe novel smaller transcript fragments. No exon 52 skipping was observedin non-treated cells (NT).

DMD Exon 53 Skipping

A series of AONs targeting sequences within exon 53 were designed andtransfected in healthy control myotube cultures. Subsequent RT-PCR andsequence analysis of isolated RNA demonstrated that almost all AONstargeting a continuous nucleotide stretch within exon 53 herein definedas SEQ ID NO 7, was indeed capable of inducing exon 53 skipping. The AONwith SEQ ID NO 328 reproducibly induced highest levels of exon 53skipping (not shown).

SEQUENCE LISTING: DMD GENE AMINO ACID SEQUENCE SEO ID NO 1:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKEGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSEGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTVTTREQILVKHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNFSDLKEKVNAIEREKAEKERKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIEFCQLLSERLNWLEYQNNIIAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDEVNRLSGLQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKELQTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNKLRKIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKGGLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESVNSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSIQSAQETEKSLHLIQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQRLQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWGKATQKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRKLVEPQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNALKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVQETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEARTIITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNT PGKPMREDTMSEQ ID NO 2 (EXON 43): AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAAGAAGACAG CAGCAUUGCAMGUGCAACGCCUGUGG SEQ ID NO 3 (EXON 46):UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCUGGAMAGAGCAGCAACUAAAAGAMAGC SEQ ID NO 4 (EXON 50):GGCGGUAMCCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUGACCUAG CUCCUGGACUGACCACUAUUGGSEQ ID NO 5 (EXON 51): CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAGGAMCUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUGUUGGAGGUAC SEQ ID NO 6 (EXON 52):AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACCGCUGCCCAAAAUUU GAAAAACAAGACCAGCAAUCAAGAGGCU SEQ ID NO 7 (EXON 53):AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUGAGCAGGUCUUAGGA CAGGCCAGAG

TABLE 1 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 43 SEQ IDCCACAGGCGUUGCACUUUGCA NO 8 AUGC SEQ ID CACAGGCGUUGCACUUUGCAA NO 9 UGCUSEQ ID ACAGGCGUUGCACUUUGCAAU NO 10 GCUG SEQ ID CAGGCGUUGCACUUUGCAAUGNO 11 CUGC SEQ ID AGGCGUUGCACULTUGCAAUGC NO 12 UGCU SEQ IDGGCGUUGCACULTUGCAAUGCU NO 13 GCUG SEQ ID GCGUUGCACULTUGCAAUGCUG NO 14CUGU SEQ ID CGUUGCACUUUGCAAUGCUGC NO 15 UGUC SEQ IDCGUUGCACULTUGCAAUGCUGC NO 16 UG PS240 SEQ ID GUUGCACUUUGCAAUGCUGCU NO 17GUCU SEQ ID UUGCACUUUGCAAUGCUGCUG NO 18 UCUU SEQ IDUGCACUUUGCAAUGCUGCUGU NO 19 CUUC SEQ ID GCACUUUGCAAUGCUGCUGUC NO 20 UUCUSEQ ID CACUUUGCAAUGCUGCUGUCU NO 21 UCUU SEQ ID ACUUUGCAAUGCUGCUGUCUUNO 22 CUUG SEQ ID CUUUGCAAUGCUGCUGUCUUC NO 23 UUGC SEQ IDUUUGCAAUGCUGCUGUCUUCU NO 24 UGCU SEQ ID UUGCAAUGCUGCUGUCUUCUU NO 25 GCUASEQ ID UGCAAUGCUGCUGUCUUCUUG NO 26 CUAU SEQ ID GCAAUGCUGCUGUCUUCUUGCNO 27 UAUG SEQ ID CAAUGCUGCUGUCUUCUUGCU NO 28 AUGA SEQ IDAAUGCUGCUGUCUUCUUGCUA NO 29 UGAA SEQ ID AUGCUGCUGUCUUCUUGCUAU NO 30 GAAUSEQ ID UGCUGCUGUCUUCUUGCUAUG NO 31 AAUA SEQ ID GCUGCUGUCUUCUUGCUAUGANO 32 AUAA SEQ ID CUGCUGUCUUCUUGCUAUGAA NO 33 UAAU SEQ IDUGCUGUCUUCUUGCUAUGAAU NO 34 AAUG SEQ ID GCUGUCUUCUUGCUAUGAAUA NO 35 AUGUSEQ ID CUGUCUUCUUGCUAUGAAUAA NO 36 UGUC SEQ ID UGUCUUCUUGCUAUGAAUAAUNO 37 GUCA SEQ ID GUCUUCUUGCUAUGAAUAAUG NO 38 UCAA SEQ IDUCUUCUUGCUAUGAAUAAUGUC NO 39 AAU SEQ ID CUUCUUGCUAUGAAUAAUGUCA NO 40 AUCSEQ ID UUCUUGCUAUGAAUAAUGUCAA NO 41 UCC SEQ ID UCUUGCUAUGAAUAAUGUCAAUNO 42 CCG SEQ ID CUUGCUAUGAAUAAUGUCAAUC NO 43 CGA SEQ IDUUGCUAUGAAUAAUGUCAAUCC NO 44 GAC SEQ ID UGCUAUGAAUAAUGUCAAUCCG NO 45 ACCSEQ ID GCUAUGAAUAAUGUCAAUCCGA NO 46 CCU SEQ ID CUAUGAAUAAUGUCAAUCCGACCNO 47 UG SEQ ID UAUGAAUAAUGUCAAUCCGACC NO 48 UGA SEQ IDAUGAAUAAUGUCAAUCCGACCU NO 49 GAG SEQ ID UGAAUAAUGUCAAUCCGACCUG NO 50 AGCSEQ ID GAAUAAUGUCAAUCCGACCUGA NO 51 GCU SEQ ID AAUAAUGUCAAUCCGACCUGAGCNO 52 UU SEQ ID AUAAUGUCAAUCCGACCUGAGCU NO 53 UU SEQ IDUAAUGUCAAUCCGACCUGAGCUU NO 54 UG SEQ ID AAUGUCAAUCCGACCUGAGCUUU NO 55 GUSEQ ID AUGUCAAUCCGACCUGAGCUUUG NO 56 UU SEQ ID UGUCAAUCCGACCUGAGCUUUGUNO 57 UG SEQ ID GUCAAUCCGACCUGAGCUUUGUU NO 58 GU SEQ IDUCAAUCCGACCUGAGCUUUGUUG NO 59 UA SEQ ID CAAUCCGACCUGAGCUUUGUUGU NO 60 AGSEQ ID AAUCCGACCUGAGCUUUGUUGU NO 61 AGA SEQ ID AUCCGACCUGAGCUUUGUUGUANO 62 GAC SEQ ID UCCGACCUGAGCUUUGUUGUAG NO 63 ACU SEQ IDCCGACCUGAGCUUUGUUGUAGAC NO 64 UA SEQ ID CGACCUGAGCUUUGUUGUAG NO 65 PS237SEQ ID CGACCUGAGCUUUGUUGUAGAC NO 66 UAU PS238 SEQ IDGACCUGAGCUUUGUUGUAGACU NO 67 AUC SEQ ID ACCUGAGCUUUGUUGUAGACUA NO 68 UCASEQ ID CCUGAGCUUUGUUGUAGACU NO 69 AUC

TABLE 2 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 46 SEQ IDGCUUUUCUUUUAGUUGCUGCUC NO 70 UUU PS179 SEQ ID CUUUUCUUUUAGUUGCUGCUCUNO 71 UUU SEQ ID UUUUCUUUUAGUUGCUGCUCU NO 72 UUUC SEQ IDUUUCUUUUAGUUGCUGCUCUU NO 73 UUCC SEQ ID UUCUUUUAGUUGCUGCUCUUU NO 74 UCCASEQ ID UCUUUUAGUUGCUGCUCUUUUC NO 75 CAG SEQ ID CUUUUAGUUGCUGCUCUUUUCCNO 76 AGG SEQ ID UUUUAGUUGCUGCUCUUUUCCA NO 77 GGU SEQ IDUUUAGUUGCUGCUCUUUUCCAG NO 78 GUU SEQ ID UUAGUUGCUGCUCUUUUCCAGG NO 79 UUCSEQ ID UAGUUGCUGCUCUUUUCCAGGU NO 80 UCA SEQ ID AGUUGCUGCUCUUUUCCAGGUUNO 81  CAA SEQ ID GUUGCUGCUCUUUUCCAGGUUC NO 82 AAG SEQ IDUUGCUGCUCUUUUCCAGGUUCA NO 83 AGU SEQ ID UGCUGCUCUUUUCCAGGUUCAA NO 84 GUGSEQ ID GCUGCUCUUUUCCAGGUUCAAG NO 85 UGG SEQ ID CUGCUCUUUUCCAGGUUCAAGUNO 86 GGG SEQ ID UGCUCUUUUCCAGGUUCAAGUG NO 87 GGA SEQ IDGCUCUUUUCCAGGUUCAAGUGG NO 88 GAC SEQ ID CUCUUUUCCAGGUUCAAGUGGG NO 89 AUASEQ ID UCUUUUCCAGGUUCAAGUGGG NO 90 AUAC SEQ ID UCUUUUCCAGGUUCAAGUGGNO 91 PS177 SEQ ID CUUUUCCAGGUUCAAGUGGGA NO 92 UACU SEQ IDUUUUCCAGGUUCAAGUGGGAU NO 93 ACUA SEQ ID UUUCCAGGUUCAAGUGGGAUA NO 94 CUAGSEQ ID UUCCAGGUUCAAGUGGGAUAC NO 95 UAGC SEQ ID UCCAGGUUCAAGUGGGAUACUNO 96 AGCA SEQ ID CCAGGUUCAAGUGGGAUACUA NO 97 GCAA SEQ IDCAGGUUCAAGUGGGAUACUAG NO 98 CAAU SEQ ID AGGUUCAAGUGGGAUACUAGC NO 99 AAUGSEQ ID GGUUCAAGUGGGAUACUAGCA NO 100 AUGU SEQ ID GUUCAAGUGGGAUACUAGCAANO 101 UGUU SEQ ID UUCAAGUGGGAUACUAGCAAU NO 102 GUUA SEQ IDUCAAGUGGGAUACUAGCAAUG NO 103 UUAU SEQ ID CAAGUGGGAUACUAGCAAUGU NO 104UAUC SEQ ID AAGUGGGAUACUAGCAAUGUU NO 105 AUCU SEQ IDAGUGGGAUACUAGCAAUGUUA NO 106 UCUG SEQ ID GUGGGAUACUAGCAAUGUUAU NO 107CUGC SEQ ID UGGGAUACUAGCAAUGUUAUC NO 108 UGCU SEQ IDGGGAUACUAGCAAUGUUAUCU NO 109 GCUU SEQ ID GGAUACUAGCAAUGUUAUCUG NO 110CUUC PS181 SEQ ID GAUACUAGCAAUGUUAUCUGC NO 111 UUCC SEQ IDAUACUAGCAAUGUUAUCUGCU NO 112 UCCU SEQ ID UACUAGCAAUGUUAUCUGCUU NO 113CCUC SEQ ID ACUAGCAAUGUUAUCUGCUUCC NO 114 UCC SEQ IDCUAGCAAUGUUAUCUGCUUCCU NO 115 CCA SEQ ID UAGCAAUGUUAUCUGCUUCCUC NO 116CAA SEQ ID AGCAAUGUUAUCUGCUUCCUCC NO 117 AAC PS182 SEQ IDGCAAUGUUAUCUGCUUCCUCCA NO 118 ACC SEQ ID CAAUGUUAUCUGCUUCCUCCAA NO 119CCA SEQ ID AAUGUUAUCUGCUUCCUCCAAC NO 120 CAU SEQ IDAUGUUAUCUGCUUCCUCCAACC NO 121 AUA SEQ ID UGUUAUCUGCUUCCUCCAACCA NO 122UAA

TABLE 3 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 50 SEQ IDCCAAUAGUGGUCAGUCCAGGA NO 123 GCUA SEQ ID CAAUAGUGGUCAGUCCAGGAG NO 124CUAG SEQ ID AAUAGUGGUCAGUCCAGGAGC NO 125 UAGG SEQ IDAUAGUGGUCAGUCCAGGAGCU NO 126 AGGU SEQ ID AUAGUGGUCAGUCCAGGAGCU NO 127PS248 SEQ ID UAGUGGUCAGUCCAGGAGCUA NO 128 GGUC SEQ IDAGUGGUCAGUCCAGGAGCUAG NO 129 GUCA SEQ ID GUGGUCAGUCCAGGAGCUAGG NO 130UCAG SEQ ID UGGUCAGUCCAGGAGCUAGGU NO 131 CAGG SEQ IDGGUCAGUCCAGGAGCUAGGUC NO 132 AGGC SEQ ID GUCAGUCCAGGAGCUAGGUCA NO 133GGCU SEQ ID UCAGUCCAGGAGCUAGGUCAG NO 134 GCUG SEQ IDCAGUCCAGGAGCUAGGUCAGG NO 135 CUGC SEQ ID AGUCCAGGAGCUAGGUCAGGC NO 136UGCU SEQ ID GUCCAGGAGCUAGGUCAGGCU NO 137 GCUU SEQ IDUCCAGGAGCUAGGUCAGGCUG NO 138 CUUU SEQ ID CCAGGAGCUAGGUCAGGCUGC NO 139UUUG SEQ ID  CAGGAGCUAGGUCAGGCUGCU NO 140 UUGC SEQ IDAGGAGCUAGGUCAGGCUGCUU NO 141 UGCC SEQ ID GGAGCUAGGUCAGGCUGCUUU NO 142GCCC SEQ ID GAGCUAGGUCAGGCUGCUUUG NO 143 CCCU SEQ IDAGCUAGGUCAGGCUGCUUUGC NO 144 CCUC SEQ ID GCUAGGUCAGGCUGCUUUGCC NO 145CUCA SEQ ID CUAGGUCAGGCUGCUUUGCCCU NO 146 CAG SEQ IDUAGGUCAGGCUGCUUUGCCCUC NO 147 AGC SEQ ID AGGUCAGGCUGCUUUGCCCUCA NO 148GCU SEQ ID GGUCAGGCUGCUUUGCCCUCAG NO 149 CUC SEQ IDGUCAGGCUGCUUUGCCCUCAGC NO 150 UCU SEQ ID UCAGGCUGCUUUGCCCUCAGCU NO 151CUU SEQ ID CAGGCUGCUUUGCCCUCAGCUC NO 152 UUG SEQ IDAGGCUGCUUUGCCCUCAGCUCU NO 153 UGA SEQ ID GGCUGCUUUGCCCUCAGCUCUU NO 154GAA SEQ ID GCUGCUUUGCCCUCAGCUCUUG NO 155 AAG SEQ IDCUGCUUUGCCCUCAGCUCUUGA NO 156 AGU SEQ ID UGCUUUGCCCUCAGCUCUUGAA NO 157GUA SEQ ID GCUUUGCCCUCAGCUCUUGAAG NO 158 UAA SEQ IDCUUUGCCCUCAGCUCUUGAAGU NO 159 AAA SEQ ID UUUGCCCUCAGCUCUUGAAGU NO 160AAAC SEQ ID UUGCCCUCAGCUCUUGAAGUA NO 161 AACG SEQ IDUGCCCUCAGCUCUUGAAGUAA NO 162 ACGG SEQ ID GCCCUCAGCUCUUGAAGUAAAC NO 163GGU SEQ ID CCCUCAGCUCUUGAAGUAAACG NO 164 GUU SEQ ID CCUCAGCUCUUGAAGUAAACNO 165 PS246 SEQ ID CCUCAGCUCUUGAAGUAAACG NO 166 PS247 SEQ IDCUCAGCUCUUGAAGUAAACG NO 167 PS245 SEQ ID CCUCAGCUCUUGAAGUAAACG NO 529GUUU SEQ ID CUCAGCUCUUGAAGUAAACGG NO 530 UUUA SEQ IDUCAGCUCUUGAAGUAAACGGU NO 531 UUAC SEQ ID CAGCUCUUGAAGUAAACGGUU NO 532UACC SEQ ID AGCUCUUGAAGUAAACGGUUU NO 533 ACCG SEQ IDGCUCUUGAAGUAAACGGUUUA NO 534 CCGC SEQ ID CUCUUGAAGUAAACGGUUUAC NO 535CGCC

TABLE 4 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 51 SEQ IDGUACCUCCAACAUCAAGGAAGA NO 168 UGG SEQ ID UACCUCCAACAUCAAGGAAGAU NO 169GGC SEQ ID ACCUCCAACAUCAAGGAAGAUG NO 170 GCA SEQ IDCCUCCAACAUCAAGGAAGAUGG NO 171 CAU SEQ ID CUCCAACAUCAAGGAAGAUGGC NO 172AUU SEQ ID UCCAACAUCAAGGAAGAUGGCA NO 173 UUU SEQ IDCCAACAUCAAGGAAGAUGGCAU NO 174 UUC SEQ ID CAACAUCAAGGAAGAUGGCAUU NO 175UCU SEQ ID AACAUCAAGGAAGAUGGCAUUU NO 176 CUA SEQ IDACAUCAAGGAAGAUGGCAUUUC NO 177 UAG SEQ ID CAUCAAGGAAGAUGGCAUUUCU NO 178AGU SEQ ID AUCAAGGAAGAUGGCAUUUCUA NO 179 GUU SEQ IDUCAAGGAAGAUGGCAUUUCUAG NO 180 UUU SEQ ID CAAGGAAGAUGGCAUUUCUAGU NO 181UUG SEQ ID AAGGAAGAUGGCAUUUCUAGUU NO 182 UGG SEQ IDAGGAAGAUGGCAUUUCUAGUUU NO 183 GGA SEQ ID GGAAGAUGGCAUUUCUAGUUUG NO 184GAG SEQ ID GAAGAUGGCAUUUCUAGUUUGG NO 185 AGA SEQ IDAAGAUGGCAUUUCUAGUUUGGA NO 186 GAU SEQ ID AGAUGGCAUUUCUAGUUUGGAG NO 187AUG SEQ ID GAUGGCAUUUCUAGUUUGGAGA NO 188 UGG SEQ IDAUGGCAUUUCUAGUUUGGAGAU NO 189 GGC SEQ ID UGGCAUUUCUAGUUUGGAGAUG NO 190GCA SEQ ID GGCAUUUCUAGUUUGGAGAUGG NO 191 CAG SEQ IDGCAUUUCUAGUUUGGAGAUGGC NO 192 AGU SEQ ID CAUUUCUAGUUUGGAGAUGGCA NO 193GUU SEQ ID AUUUCUAGUUUGGAGAUGGCAG NO 194 UUU SEQ IDUUUCUAGUUUGGAGAUGGCAGU NO 195 UUC SEQ ID UUCUAGUUUGGAGAUGGCAGUU NO 196UCC SEQ ID UCUAGUUUGGAGAUGGCAGUUU NO 197 CCU SEQ IDCUAGUUUGGAGAUGGCAGUUUC NO 198 CUU SEQ ID UAGUUUGGAGAUGGCAGUUUCC NO 199UUA SEQ ID AGUUUGGAGAUGGCAGUUUCCU NO 200 UAG SEQ IDGUUUGGAGAUGGCAGUUUCCUU NO 201 AGU SEQ ID UUUGGAGAUGGCAGUUUCCUUA NO 202GUA SEQ ID UUGGAGAUGGCAGUUUCCUUAG NO 203 UAA SEQ IDUGGAGAUGGCAGUUUCCUUAGU NO 204 AAC SEQ ID GAGAUGGCAGUUUCCUUAGUAA NO 205CCA SEQ ID AGAUGGCAGUUUCCUUAGUAAC NO 206 CAC SEQ IDGAUGGCAGUUUCCUUAGUAACC NO 207 ACA SEQ ID AUGGCAGUUUCCUUAGUAACCA NO 208CAG SEQ ID UGGCAGUUUCCUUAGUAACCAC NO 209 AGG SEQ IDGGCAGUUUCCUUAGUAACCACA NO 210 GGU SEQ ID GCAGUUUCCUUAGUAACCACAG NO 211GUU SEQ ID CAGUUUCCUUAGUAACCACAGG NO 212 UUG SEQ IDAGUUUCCUUAGUAACCACAGGU NO 213 UGU SEQ ID GUUUCCUUAGUAACCACAGGUU NO 214GUG SEQ ID UUUCCUUAGUAACCACAGGUUG NO 215 UGU SEQ IDUUCCUUAGUAACCACAGGUUGU NO 216 GUC SEQ ID UCCUUAGUAACCACAGGUUGUG NO 217UCA SEQ ID CCUUAGUAACCACAGGUUGUGU NO 218 CAC SEQ IDCUUAGUAACCACAGGUUGUGUC NO 219 ACC SEQ ID UUAGUAACCACAGGUUGUGUCA NO 220CCA SEQ ID UAGUAACCACAGGUUGUGUCAC NO 221 CAG SEQ IDAGUAACCACAGGUUGUGUCACC NO 222 AGA SEQ ID GUAACCACAGGUUGUGUCACCA NO 223GAG SEQ ID UAACCACAGGUUGUGUCACCAG NO 224 AGU SEQ IDAACCACAGGUUGUGUCACCAGA NO 225 GUA SEQ ID ACCACAGGUUGUGUCACCAGAG NO 226UAA SEQ ID CCACAGGUUGUGUCACCAGAGU NO 227 AAC SEQ IDCACAGGUUGUGUCACCAGAGUA NO 228 ACA SEQ ID ACAGGUUGUGUCACCAGAGUAA NO 229CAG SEQ ID CAGGUUGUGUCACCAGAGUAAC NO 230 AGU SEQ IDAGGUUGUGUCACCAGAGUAACA NO 231 GUC SEQ ID GGUUGUGUCACCAGAGUAACAG NO 232UCU SEQ ID GUUGUGUCACCAGAGUAACAGU NO 233 CUG SEQ IDUUGUGUCACCAGAGUAACAGUC NO 234 UGA SEQ ID UGUGUCACCAGAGUAACAGUCU NO 235GAG SEQ ID GUGUCACCAGAGUAACAGUCUG NO 236 AGU SEQ IDUGUCACCAGAGUAACAGUCUGA NO 237 GUA SEQ ID GUCACCAGAGUAACAGUCUGAG NO 238UAG SEQ ID UCACCAGAGUAACAGUCUGAGU NO 239 AGG SEQ IDCACCAGAGUAACAGUCUGAGUA NO 240 GGA SEQ ID ACCAGAGUAACAGUCUGAGUA NO 241GGAG

TABLE 5 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 52 SEQ IDAGCCUCUUGAUUGCUGGUCUUG NO 242 UUU SEQ ID GCCUCUUGAUUGCUGGUCUUGU NO 243UUU SEQ ID CCUCUUGAUUGCUGGUCUUGUU NO 244 UUU SEQ ID CCUCUUGAUUGCUGGUCUUGNO 245 SEQ ID CUCUUGAUUGCUGGUCUUGUU NO 246 UUUC PS232 SEQ IDUCUUGAUUGCUGGUCUUGUUU NO 247 UUCA SEQ ID CUUGAUUGCUGGUCUUGUUUU NO 248UCAA SEQ ID UUGAUUGCUGGUCUUGUUUUU NO 249 CAAA SEQ IDUGAUUGCUGGUCUUGUUUUUC NO 250 AAAU SEQ ID GAUUGCUGGUCUUGUUUUUCA NO 251AAUU SEQ ID GAUUGCUGGUCUUGUUUUUC NO 252 SEQ ID AUUGCUGGUCUUGUUUUUCAANO 253 AUUU SEQ ID UUGCUGGUCUUGUUUUUCAAA NO 254 UUUU SEQ IDUGCUGGUCUUGUUUUUCAAAU NO 255 UUUG SEQ ID GCUGGUCUUGUUUUUCAAAUU NO 256UUGG SEQ ID CUGGUCUUGUUUUUCAAAUUU NO 257 UGGG SEQ IDUGGUCUUGUUUUUCAAAUUUU NO 258 GGGC SEQ ID GGUCUUGUUUUUCAAAUUUUG NO 259GGCA SEQ ID GUCUUGUUUUUCAAAUUUUGG NO 260 GCAG SEQ IDUCUUGUUUUUCAAAUUUUGGG NO 261 CAGC SEQ ID CUUGUUUUUCAAAUUUUGGGC NO 262AGCG SEQ ID UUGUUUUUCAAAUUUUGGGCA NO 263 GCGG SEQ IDUGUUUUUCAAAUUUUGGGCAG NO 264 CGGU SEQ ID GUUUUUCAAAUUUUGGGCAGC NO 265GGUA SEQ ID UUUUUCAAAUUUUGGGCAGCG NO 266 GUAA SEQ IDUUUUCAAAUUUUGGGCAGCGG NO 267 UAAU SEQ ID UUUCAAAUUUUGGGCAGCGGU NO 268AAUG SEQ ID UUCAAAUUUUGGGCAGCGGUA NO 269 AUGA SEQ IDUCAAAUUUUGGGCAGCGGUAA NO 270 UGAG SEQ ID CAAAUUUUGGGCAGCGGUAAU NO 271GAGU SEQ ID AAAUUUUGGGCAGCGGUAAUG NO 272 AGUU SEQ IDAAUUUUGGGCAGCGGUAAUGA NO 273 GUUC SEQ ID AUUUUGGGCAGCGGUAAUGAG NO 274UUCU SEQ ID UUUUGGGCAGCGGUAAUGAGU NO 275 UCUU SEQ IDUUUGGGCAGCGGUAAUGAGUU NO 276 CUUC SEQ ID UUGGGCAGCGGUAAUGAGUUCU NO 277UCC SEQ ID UGGGCAGCGGUAAUGAGUUCUU NO 278 CCA SEQ IDGGGCAGCGGUAAUGAGUUCUUC NO 279 CAA SEQ ID GGCAGCGGUAAUGAGUUCUUCC NO 280AAC SEQ ID GCAGCGGUAAUGAGUUCUUCCA NO 281 ACU SEQ IDCAGCGGUAAUGAGUUCUUCCAA NO 282 CUG SEQ ID AGCGGUAAUGAGUUCUUCCAAC NO 283UGG SEQ ID GCGGUAAUGAGUUCUUCCAACU NO 284 GGG SEQ IDCGGUAAUGAGUUCUUCCAACUG NO 285 GGG SEQ ID GGUAAUGAGUUCUUCCAACUGG NO 286GGA SEQ ID GGUAAUGAGUUCUUCCAACUGG NO 287 SEQ ID GUAAUGAGUUCUUCCAACUGGGNO 288 GAC SEQ ID UAAUGAGUUCUUCCAACUGGGG NO 289 ACG SEQ IDAAUGAGUUCUUCCAACUGGGGA NO 290 CGC SEQ ID AUGAGUUCUUCCAACUGGGGAC NO 291GCC SEQ ID UGAGUUCUUCCAACUGGGGACG NO 292 CCU SEQ IDGAGUUCUUCCAACUGGGGACGC NO 293 CUC SEQ ID AGUUCUUCCAACUGGGGACGCC NO 294UCU SEQ ID GUUCUUCCAACUGGGGACGCCU NO 295 CUG SEQ IDUUCUUCCAACUGGGGACGCCUC NO 296 UGU SEQ ID UCUUCCAACUGGGGACGCCUCU NO 297GUU SEQ ID CUUCCAACUGGGGACGCCUCUG NO 298 UUC SEQ IDUUCCAACUGGGGACGCCUCUGU NO 299 UCC PS236 SEQ ID UCCAACUGGGGACGCCUCUGUUNO 300 CCA SEQ ID CCAACUGGGGACGCCUCUGUUC NO 301 CAA SEQ IDCAACUGGGGACGCCUCUGUUCC NO 302 AAA SEQ ID AACUGGGGACGCCUCUGUUCCA NO 303AAU SEQ ID ACUGGGGACGCCUCUGUUCCAA NO 304 AUC SEQ IDCUGGGGACGCCUCUGUUCCAAA NO 305 UCC SEQ ID UGGGGACGCCUCUGUUCCAAAU NO 306CCU SEQ ID GGGGACGCCUCUGUUCCAAAUC NO 307 CUG SEQ IDGGGACGCCUCUGUUCCAAAUCC NO 308 UGC SEQ ID GGACGCCUCUGUUCCAAAUCCU NO 309GCA SEQ ID GACGCCUCUGUUCCAAAUCCUG NO 310 CAU

TABLE 6 OLIGONUCLEOTIDES FOR SKIPPING DMD GENE EXON 53 SEQ IDCUCUGGCCUGUCCUAAGACCU NO 311 GCUC SEQ ID UCUGGCCUGUCCUAAGACCUG NO 312CUCA SEQ ID CUGGCCUGUCCUAAGACCUGC NO 313 UCAG SEQ IDUGGCCUGUCCUAAGACCUGCU NO 314 CAGC SEQ ID GGCCUGUCCUAAGACCUGCUC NO 315AGCU SEQ ID GCCUGUCCUAAGACCUGCUCA NO 316 GCUU SEQ IDCCUGUCCUAAGACCUGCUCAG NO 317 CUUC SEQ ID CUGUCCUAAGACCUGCUCAGC NO 318UUCU SEQ ID UGUCCUAAGACCUGCUCAGCU NO 319 UCUU SEQ IDGUCCUAAGACCUGCUCAGCUU NO 320 CUUC SEQ ID UCCUAAGACCUGCUCAGCUUC NO 321UUCC SEQ ID CCUAAGACCUGCUCAGCUUCU NO 322 UCCU SEQ IDCUAAGACCUGCUCAGCUUCUU NO 323 CCUU SEQ ID UAAGACCUGCUCAGCUUCUUC NO 324CUUA SEQ ID AAGACCUGCUCAGCUUCUUCC NO 325 UUAG SEQ IDAGACCUGCUCAGCUUCUUCCU NO 326 UAGC SEQ ID GACCUGCUCAGCUUCUUCCUU NO 327AGCU SEQ ID ACCUGCUCAGCUUCUUCCUUA NO 328 GCUU SEQ IDCCUGCUCAGCUUCUUCCUUAG NO 329 CUUC SEQ ID CUGCUCAGCUUCUUCCUUAGC NO 330UUCC SEQ ID UGCUCAGCUUCUUCCUUAGCU NO 331 UCCA SEQ IDGCUCAGCUUCUUCCUUAGCUU NO 332 CCAG SEQ ID CUCAGCUUCUUCCUUAGCUUC NO 333CAGC SEQ ID UCAGCUUCUUCCUUAGCUUCC NO 334 AGCC SEQ IDCAGCUUCUUCCUUAGCUUCCAG NO 335 CCA SEQ ID AGCUUCUUCCUUAGCUUCCAGC NO 336CAU SEQ ID GCUUCUUCCUUAGCUUCCAGCC NO 337 AUU SEQ IDCUUCUUCCUUAGCUUCCAGCCA NO 338 UUG SEQ ID UUCUUCCUUAGCUUCCAGCCAU NO 339UGU SEQ ID UCUUCCUUAGCUUCCAGCCAUU NO 340 GUG SEQ IDCUUCCUUAGCUUCCAGCCAUUG NO 341 UGU SEQ ID UUCCUUAGCUUCCAGCCAUUGU NO 342GUU SEQ ID UCCUUAGCUUCCAGCCAUUGUG NO 343 UUG SEQ IDCCUUAGCUUCCAGCCAUUGUGU NO 344 UGA SEQ ID CUUAGCUUCCAGCCAUUGUGUU NO 345GAA SEQ ID UUAGCUUCCAGCCAUUGUGUUG NO 346 AAU SEQ IDUAGCUUCCAGCCAUUGUGUUGA NO 347 AUC SEQ ID AGCUUCCAGCCAUUGUGUUGAA NO 348UCC SEQ ID GCUUCCAGCCAUUGUGUUGAAU NO 349 CCU SEQ IDCUUCCAGCCAUUGUGUUGAAUC NO 350 CUU SEQ ID UUCCAGCCAUUGUGUUGAAUCC NO 351UUU SEQ ID UCCAGCCAUUGUGUUGAAUCCU NO 352 UUA SEQ IDCCAGCCAUUGUGUUGAAUCCUU NO 353 UAA SEQ ID CAGCCAUUGUGUUGAAUCCUUU NO 354AAC SEQ ID AGCCAUUGUGUUGAAUCCUUUA NO 355 ACA SEQ IDGCCAUUGUGUUGAAUCCUUUAA NO 356 CAU SEQ ID CCAUUGUGUUGAAUCCUUUAAC NO 357AUU SEQ ID CAUUGUGUUGAAUCCUUUAACA NO 358 UUU

TABLE 7 OLIGONUCLEOTIDES FOR SKIPPING OTHEREXONS OF THE DMD GENE AS IDENTIFIED DMD Gene Exon 6 SEQ IDCAUUUUUGACCUACAUGUGG NO 359 SEQ ID UUUGACCUACAUGUGGAAAG NO 360 SEQ IDUACAUUUUUGACCUACAUGUG NO 361 GAAA G SEQ ID GGUCUCCUUACCUAUGA NO 362SEQ ID UCUUACCUAUGACUAUGGAUG NO 363 AGA SEQ ID AUUUUUGACCUACAUGGGAAANO 364 G SEQ ID UACGAGUUGAUUGUCGGACCCA NO 365 G SEQ IDGUGGUCUCCUUACCUAUGACUG NO 366 UGG SEQ ID UGUCUCAGUAAUCUUCUUACCU NO 367AU DMD Gene Exon 7 SEQ ID UGCAUGUUCCAGUCGUUGUGU NO 368 GG SEQ IDCACUAUUCCAGUCAAAUAGGU NO 369 CUGG SEQ ID AUUUACCAACCUUCAGGAUCGA NO 370GUA SEQ ID GGCCUAAAACACAUACACAUA NO 371 DMD Gene Exon 11 SEQ IDCCCUGAGGCAUUCCCAUCUUG NO 372 AAU SEQ ID AGGACUUACUUGCUUUGUUU NO 373SEQ ID CUUGAAUUUAGGAGAUUCAUCU NO 374 G SEQ ID CAUCUUCUGAUAAUUUUCCUGUNO 375 U DMD Gene Exon 17 SEQ ID CCAUUACAGUUGUCUGUGUU NO 376 SEQ IDUGACAGCCUGUGAAAUCUGUG NO 377 AG SEQ ID UAAUCUGCCUCUUCUUUUGG NO 378DMD Gene Exon 19 SEQ ID CAGCAGUAGUUGUCAUCUGC NO 379 SEQ IDGCCUGAGCUGAUCUGCUGGCA NO 380 UCUUGC SEQ ID GCCUGAGCUGAUCUGCUGGCAU NO 381CUUGCA GUU SEQ ID UCUGCUGGCAUCUUGC NO 382 DMD Gene Exon 21 SEQ IDGCCGGUUGACUUCAUCCUGUG NO 383 C SEQ ID GUCUGCAUCCAGGAACAUGGG NO 384 UCSEQ ID UACUUACUGUCUGUAGCUCUU NO 385 UCU SEQ ID CUGCAUCCAGGAACAUGGGUCCNO 386 SEQ ID GUUGAAGAUCUGAUAGCCGGUU NO 387 GA DMD Gene Exon 44 SEQ IDUCAGCUUCUGUUAGCCACUG NO 388 SEQ ID UUCAGCUUCUGUUAGCCACU NO 389 SEQ IDUUCAGCUUCUGUUAGCCACUG NO 390 SEQ ID UCAGCUUCUGUUAGCCACUGA NO 391 SEQ IDUUCAGCUUCUGUUAGCCACUG NO 392 A SEQ ID UCAGCUUCUGUUAGCCACUGA NO 393SEQ ID UUCAGCUUCUGUUAGCCACUG NO 394 A SEQ ID UCAGCUUCUGUUAGCCACUGANO 395 U SEQ ID UUCAGCUUCUGUUAGCCACUG NO 396 AU SEQ IDUCAGCUUCUGUUAGCCACUGA NO 397 UU SEQ ID UUCAGCUUCUGUUAGCCACUG NO 398 AUUSEQ ID UCAGCUUCUGUUAGCCACUGA NO 399 UUA SEQ ID UUCAGCUUCUGUUAGCCACUGNO 400 AUA SEQ ID UCAGCUUCUGUUAGCCACUGA NO 401 UUAA SEQ IDUUCAGCUUCUGUUAGCCACUG NO 402 AUUAA SEQ ID UCAGCUUCUGUUAGCCACUGA NO 403UUAAA SEQ ID UUCAGCUUCUGUUAGCCACUG NO 404 AUUAAA SEQ IDCAGCUUCUGUUAGCCACUG NO 405 SEQ ID CAGCUUCUGUUAGCCACUGAU NO 406 SEQ IDAGCUUCUGUUAGCCACUGAUU NO 407 SEQ ID CAGCUUCUGUUAGCCACUGAU NO 408 USEQ ID AGCUUCUGUUAGCCACUGAUU NO 409 A SEQ ID CAGCUUCUGUUAGCCACUGAUNO 410 UA SEQ ID AGCUUCUGUUAGCCACUGAUU NO 411 AA SEQ IDCAGCUUCUGUUAGCCACUGAU NO 412 UAA SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 413 AASEQ ID CAGCUUCUGUUAGCCACUGAUU NO 414 AAA SEQ ID AGCUUCUGUUAGCCACUGAUUANO 415 AA SEQ ID AGCUUCUGUUAGCCACUGAU NO 416 SEQ ID GCUUCUGUUAGCCACUGAUUNO 417 SEQ ID AGCUUCUGUUAGCCACUGAUU NO 418 SEQ ID GCUUCUGUUAGCCACUGAUUANO 419 SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 420 SEQ IDGCUUCUGUUAGCCACUGAUUAA NO 421 SEQ ID AGCUUCUGUUAGCCACUGAUUA NO 422 ASEQ ID GCUUCUGUUAGCCACUGAUUAA NO 423 A SEQ ID AGCUUCUGUUAGCCACUGAUUANO 424 AA SEQ ID GCUUCUGUUAGCCACUGAUUAA NO 425 A SEQ IDCCAUUUGUAUUUAGCAUGUUCC NO 426 C SEQ ID AGAUACCAUUUGUAUUUAGC NO 427SEQ ID GCCAUUUCUCAACAGAUCU NO 428 SEQ ID GCCAUUUCUCAACAGAUCUGUC NO 429 ASEQ ID AUUCUCAGGAAUUUGUGUCUUU NO 430 C SEQ ID UCUCAGGAAUUUGUGUCUUUCNO 431 SEQ ID GUUCAGCUUCUGUUAGCC NO 432 SEQ ID CUGAUUAAAUAUCUUUAUAUCNO 433 SEQ ID GCCGCCAUUUCUCAACAG NO 434 SEQ ID GUAUUUAGCAUGUUCCCA NO 435SEQ ID CAGGAAUUUGUGUCUUUC NO 436 DMD Gene Exon 45 SEQ IDUUUGCCGCUGCCCAAUGCCAU NO 437 CCUG SEQ ID AUUCAAUGUUCUGACAACAGU NO 438UUGC SEQ ID CCAGUUGCAUUCAAUGUUCUG NO 439 ACAA SEQ IDCAGUUGCAUUCAAUGUUCUGA NO 440 C SEQ ID AGUUGCAUUCAAUGUUCUGA NO 441 SEQ IDGAUUGCUGAAUUAUUUCUUCC NO 442 SEQ ID GAUUGCUGAAUUAUUUCUUCC NO 443 CCAGSEQ ID AUUGCUGAAUUAUUUCUUCCC NO 444 CAGU SEQ ID UUGCUGAAUUAUUUCUUCCCCNO 445 AGUU SEQ ID UGCUGAAUUAUUUCUUCCCCA NO 446 GUUG SEQ IDGCUGAAUUAUUUCUUCCCCAG NO 447 UUGC SEQ ID CUGAAUUAUUUCUUCCCCAGU NO 448UGCA SEQ ID UGAAUUAUUUCUUCCCCAGUU NO 449 GCAU SEQ IDGAAUUAUUUCUUCCCCAGUUG NO 450 CAUU SEQ ID AAUUAUUUCUUCCCCAGUUGC NO 451AUUC SEQ ID AUUAUUUCUUCCCCAGUUGCA NO 452 UUCA SEQ IDUUAUUUCUUCCCCAGUUGCAU NO 453 UCAA SEQ ID UAUUUCUUCCCCAGUUGCAUU NO 454CAAU SEQ ID AUUUCUUCCCCAGUUGCAUUC NO 455 AAUG SEQ IDUUUCUUCCCCAGUUGCAUUCA NO 456 AUGU SEQ ID UUCUUCCCCAGUUGCAUUCAA NO 457UGUU SEQ ID UCUUCCCCAGUUGCAUUCAAU NO 458 GUUC SEQ IDCUUCCCCAGUUGCAUUCAAUG NO 459 UUCU SEQ ID UUCCCCAGUUGCAUUCAAUGU NO 460UCUG SEQ ID UCCCCAGUUGCAUUCAAUGUU NO 461 CUGA SEQ IDCCCCAGUUGCAUUCAAUGUUC NO 462 UGAC SEQ ID CCCAGUUGCAUUCAAUGUUCU NO 463GACA SEQ ID CCAGUUGCAUUCAAUGUUCUG NO 464 ACAA SEQ IDCAGUUGCAUUCAAUGUUCUGA NO 465 CAAC SEQ ID AGUUGCAUUCAAUGUUCUGAC NO 466AACA SEQ ID UCC UGU AGA AUA CUG GCA NO 467 UC SEQ IDUGCAGACCUCCUGCCACCGCAG NO 468 AUUCA SEQ ID UUGCAGACCUCCUGCCACCGCA NO 469GAUUC AGGCUUC SEQ ID GUUGCAUUCAAUGUUCUGACAA NO 470 CAG SEQ IDUUGCAUUCAAUGUUCUGACAAC NO 471 AGU SEQ ID UGCAUUCAAUGUUCUGACAACA NO 472GUU SEQ ID GCAUUCAAUGUUCUGACAACAG NO 473 UUU SEQ IDCAUUCAAUGUUCUGACAACAGU NO 474 UUG SEQ ID AUUCAAUGUUCUGACAACAGUU NO 475UGC SEQ ID UCAAUGUUCUGACAACAGUUUG NO 476 CCG SEQ IDCAAUGUUCUGACAACAGUUUGC NO 477 CGC SEQ ID AAUGUUCUGACAACAGUUUGCC NO 478GCU SEQ ID  AUGUUCUGACAACAGUUUGCCG NO 479 CUG SEQ IDUGUUCUGACAACAGUUUGCCGC NO 480 UGC SEQ ID GUUCUGACAACAGUUUGCCGCU NO 481GCC SEQ ID UUCUGACAACAGUUUGCCGCUG NO 482 CCC SEQ IDUCUGACAACAGUUUGCCGCUGC NO 483 CCA SEQ ID CUGACAACAGUUUGCCGCUGCC NO 484CAA SEQ ID UGACAACAGUUUGCCGCUGCCC NO 485 AAU SEQ IDGACAACAGUUUGCCGCUGCCCA NO 486 AUG SEQ ID ACAACAGUUUGCCGCUGCCCAA NO 487UGC SEQ ID CAACAGUUUGCCGCUGCCCAAU NO 488 GCC SEQ IDAACAGUUUGCCGCUGCCCAAUG NO 489 CCA SEQ ID ACAGUUUGCCGCUGCCCAAUGC NO 490CAU SEQ ID CAGUUUGCCGCUGCCCAAUGCC NO 491 AUC SEQ IDAGUUUGCCGCUGCCCAAUGCCA NO 492 UCC SEQ ID GUUUGCCGCUGCCCAAUGCCAU NO 493CCU SEQ ID UUUGCCGCUGCCCAAUGCCAUC NO 494 CUG SEQ IDUUGCCGCUGCCCAAUGCCAUCC NO 495 UGG SEQ ID UGCCGCUGCCCAAUGCCAUCCU NO 496GGA SEQ ID GCCGCUGCCCAAUGCCAUCCUG NO 497 GAG SEQ IDCCGCUGCCCAAUGCCAUCCUGG NO 498 AGU SEQ ID CGCUGCCCAAUGCCAUCCUGGA NO 499GUU SEQ ID UGUUUUUGAGGAUUGCUGAA NO 500 SEQ ID UGUUCUGACAACAGUUUGCCGCNO 501 UGCCCA AUGCCAUCCUGG DMD Gene Exon 55 SEQ ID CUGUUGCAGUAAUCUAUGAGNO 502 SEQ ID UGCAGUAAUCUAUGAGUUUC NO 503 SEQ ID GAGUCUUCUAGGAGCCUUNO 504 SEQ ID UGCCAUUGUUUCAUCAGCUCUU NO 505 U SEQ IDUCCUGUAGGACAUUGGCAGU NO 506 SEQ ID CUUGGAGUCUUCUAGGAGCC NO 507DMD Gene Exon 57 SEQ ID UAGGUGCCUGCCGGCUU NO 508 SEQ IDUUCAGCUGUAGCCACACC NO 509 SEQ ID CUGAACUGCUGGAAAGUCGCC NO 510 SEQ IDCUGGCUUCCAAAUGGGACCUGA NO 511 AAAAGA AC DMD Gene Exon 59 SEQ IDCAAUUUUUCCCACUCAGUAUU NO 512 SEQ ID UUGAAGUUCCUGGAGUCUU NO 513 SEQ IDUCCUCAGGAGGCAGCUCUAAAU NO 514 DMD Gene Exon 62 SEQ ID  UGGCUCUCUCCCAGGGNO 515 SEQ ID GAGAUGGCUCUCUCCCAGGGA NO 516 CCCUGG SEQ IDGGGCACUUUGUUUGGCG NO 517 DMD Gene Exon 63 SEQ ID GGUCCCAGCAAGUUGUUUGNO 518 SEQ ID  UGGGAUGGUCCCAGCAAGUUG NO 519 UUUG SEQ IDGUAGAGCUCUGUCAUUUUGGG NO 520 DMD Gene Exon 65 SEQ IDGCUCAAGAGAUCCACUGCAAA NO 521 AAAC SEQ ID  GCCAUACGUACGUAUCAUAAA NO 522CAUUC SEQ ID UCUGCAGGAUAUCCAUGGGCUG NO 523 GUC DMD Gene Exon 66 SEQ IDGAUCCUCCCUGUUCGUCCCCUA NO 524 UUAUG DMD Gene Exon 69 SEQ IDUGCUUUAGACUCCUGUACCUG NO 525 AUA DMD Gene Exon 75 SEQ IDGGCGGCCUUUGUGUUGAC NO 526 SEQ ID GGACAGGCCUUUAUGUUCGUG NO 527 CUGCSEQ ID CCUUUAUGUUCGUGCUGCU NO 528

What is claimed is:
 1. An antisense oligonucleotide whose base sequenceconsists of 5′-CUCUUGAUUGCUGGUCUUGUUUUUC-3′ (SEQ ID NO:246), wherein theoligonucleotide comprises a modification.
 2. The antisenseoligonucleotide of claim 1, wherein the modification comprises at leastone nucleotide analogue, wherein the nucleotide analogue comprises amodified sugar moiety, a modified backbone, a modified internucleosidelinkage, or a modified base, or a combination thereof.
 3. The antisenseoligonucleotide of claim 1, wherein the modification comprises amodified sugar moiety.
 4. The antisense oligonucleotide of claim 3,wherein the modified sugar moiety is mono- or di-substituted at the 2′,3′ and/or 5′ position.
 5. The antisense oligonucleotide of claim 4,wherein the modified sugar moiety comprises a 2′-O-methyl ribose.
 6. Theantisense oligonucleotide of claim 1, wherein the modification comprisesa modified backbone.
 7. The antisense oligonucleotide of claim 6,wherein the modified backbone comprises a morpholino backbone, acarbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxidebackbone, a sulfone backbone, a formacetyl backbone, a thioformacetylbackbone, a methyleneformacetyl backbone, a riboacetyl backbone, analkene containing backbone, a sulfamate backbone, a sulfonate backbone,a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazinobackbone or an amide backbone, or a combination thereof.
 8. Theantisense oligonucleotide of claim 7, wherein the modified backbonecomprises a morpholino backbone.
 9. The antisense oligonucleotide ofclaim 1, wherein the modification comprises a modified internucleosidelinkage.
 10. The antisense oligonucleotide of claim 9, wherein themodified internucleoside linkage comprises a phosphorothioate linkage.11. The antisense oligonucleotide of claim 1, wherein the modificationcomprises a modified base.
 12. The antisense oligonucleotide of claim 1,wherein the oligonucleotide comprises a morpholino ring, aphosphorodiamidate internucleoside linkage, a peptide nucleic acid, alocked nucleic acid (LNA), or a combination thereof.
 13. The antisenseoligonucleotide of claim 1, wherein the oligonucleotide comprises a2′-O-methyl phosphorothioate ribose.
 14. The antisense oligonucleotideof claim 1, wherein the oligonucleotide comprises a phosphorodiamidatemorpholino oligomer (PMO).
 15. A pharmaceutical composition, comprisingthe antisense oligonucleotide of claim 1 and a pharmaceuticallyacceptable carrier.
 16. A method of treating Duchenne muscular dystrophyor Becker muscular dystrophy in a subject, comprising administering tothe subject the antisense oligonucleotide of claim 1.